The present invention relates to methods an apparatus for determining the concentration of tissue pigments, such as hemoglobin, using time-resolved pulses of light. The present invention relies upon the decay characteristics associated with absorptive pigments to derive their concentrations.
The utility of optical methods for studying metabolism and oxidative processes in cells and tissues was significantly enhanced in the early 1950's when time-sharing dual-wavelength systems were developed for the quantitation of small changes in the absorption of light in the visible and near-infrared (NIR) regions in highly scattering media, such as cell suspensions or muscle tissues. See Chance, B. Rev. Sci. Instrum. 22,619-627 (1951); Chance B. Biochemistry of Copper, ed. Peisach, J. (Academic, New York), pp. 293-303 (1966), which are incorporated by reference as if fully reproduced herein. Fluorescence signals from mitochondrial NADH complemented the absorption method for studies of the surface of heart, brain, and skeletal tissue. See Chance, B., et al., Nature (London) 195, 1073-1075 (1962) and Chance B. Science 120, 767-775 (1954). NIR spectroscopy has been used to detect the redox state of the copper component of cytochrome oxidase in mitochondria and yeast cells. See Chance, B. Biochemistry of Cooper, ed. Peisach, J. (Academic, New York), pp. 293-303 (1966); Chance, B. J. Biol. Chem 234, 3036-3040 (1959). Jobsis-Vander Vliet and coworkers pioneered the study of NIR absorption in tissue by transillumination. See Jobsis-Vander Vliet, F. F. Adv. Exp. Med. Biol. 191, 833-842 (1985); Jobsis-Vander Vliet, F. F., et al., J. Appl. Physiol. 113, 858,872 (1977); see also Rosenthal, M., et al., Brain Res. 108, 143-154 (1976). Algorithms have been developed to compensate for the interference from hemoglobin and myoglobin with cytochrome copper, as the latter may constitute as little as 5% of the total signal at 830 nm. See vanderZee. P. & Delpy, D. T. (1988) Oxygen Transport to Tissue X, eds. Mochizuki, M., et al., (Plenum, New York), pp. 191-197); see also Tamura, M. H., et al., Chemoreceptors and Reflexes in Breathing, ed. Lahiri, S. (Oxford, New York), in press.
An application of the principles of spectrophotometry is the use of continuous light (CW) illumination to determine the attenuation characteristic of light in systems containing localized deoxyhemoglobin (Hb) and to observe hypoxia in the brain. However, although CW systems can be used to quantify changes in optical density, they cannot quantify the concentration of an absorptive pigment.
The change in intensity of light, or the optical density (log I.sub.o /I), in an absorbing medium generally follows the Beer-Lambert Law: ##EQU1## where I is the intensity of light, E is the extinction coefficient, C is the concentration of an absorptive pigment in the medium, L is the optical path length, and the absorption per unit length is defined as u=EC=1/L (log I.sub.o /I). This law is the basis for modern spectrophotometry and, as well known to those of ordinary skill, has been repeatedly verified by both research and in industrial applications. The Beer-Lambert Law requires the determination of at least one of two quantities--specific absorption or path length--in addition to intensity. Since CW systems can only quantify changes in intensity, only the product uL can be determined.
When light is directed into an absorptive medium such as a tissue region, it is scattered, reflected, or it migrates from one point to another by a random walk or other diffusion process. The actual length traversed may be the distance between the light input and output in a rectilinear direction, since little or no light is believed to move in an orthagonal direction, except where florescence or other processes may be involved. Under these conditions, CW systems thus cannot independently quantify the specific absorption for a given medium. Therefore, the determination of the concentration of an absorptive pigment must necessarily be carried out by arbitrary calibrations using media of known values of CE in order to determine the relationship between optical density (OD) and path length (L). After L has been determined, the system can then be used to determine subsequent variations in the concentration of the absorptive pigment within a tissue region. This is the general method by which CW absorption spectrometry is carried out with scattering materials. Often, information concerning L is obtained at an adjacent wavelength, such as an isosbestic point. This method is particularly effective when two values of L are similar leading to what is termed precise dual wavelength spectrophotometry of biological tissue. See, for example, the system disclosed in U.S. patent application Ser. No. 266,166, filed Nov. 2, 1988, entitled, "Optical Coupling System For Use In Monitoring Oxygenation State Within Living Tissue", which is incorporated herein by reference a if fully reproduced herein.
Difficulties arise, however, when C for an absorptive material cannot be varied in a rational or known manner. For example, in living tissues, a known change in concentration, C, of an absorptive pigment such as hemoglobin may be difficult to impose, and if imposed, may result in traumatic consequences to the subject. In the case of limbs, tourniquet ischemia can be used to cause a known change in the concentration of hemoglobin, but individual variations and physiological adaptations may falsify any calibrations made. In the brain and heart, where oxygenation data are most needed, it is not possible to calibrate by large perturbations of the system, since damage or death might result. Under these conditions, it is difficult to calibrate the meaning of the trends in observations that may occur in some diseases such as neonatal hypoxia or adult stroke. Thus, the calibration method described loses its force and reliability. Similarly, although animal models have been resorted to for purposes of calibration, since the boundary conditions for photon migration--which are crucial--are likely to differ between the animal model and a human subject, these methods have also generally proven unsatisfactory.